Summary Decades of study have revealed that genome organization is non-random and critically impacts many nuclear processes including the regulation of transcription, DNA replication, and DNA repair, and increasing evidence suggests that the three-dimensional structures adopted by chromosomes are critical for development and are often perturbed in disease. Much of our current understanding comes from biochemical techniques performed on large populations of cells, leading to many gaps in our understanding of the mechanisms that establish and maintain organizational states, particularly in the context of individual cells. We propose to introduce a new set of single-cell technologies based on the single-molecule super-resolution imaging method DNA-PAINT to bridge this gap with a suite of tools possessing both high multiplexibility and spatial resolution. Specifically, in Aim 1 we will develop a multiplexed (>20 color) super-resolution chromosomal imaging strategy to image genomic targets ranging from kilobases to multiple megabases in Iength, which will enable us to investigate the folding properties of the chromatin fiber in single cells over a range of length-scales. In Aim 2, we will develop multiplexed assays to co-localize proteins, RNA molecules, and specific genomic sites in individual cells at the nanoscale. We will then investigate organization of architectural proteins at a model hub of large chromatin loops on the human inactive X-chromosome. In Aim 3, we will develop a proximity-dependent super-resolution method to probe specific interactions between protein and DNA targets that will allow for the sensitive detection of molecular interactions in crowded environments. We will deploy this technology to query the composition and epigenetic states of the aforementioned chromatin looping hub in individual cells. Collectively, our methods will make many questions about the positioning, composition, and epigenetic states of specific genomic loci in individual cells accessible to researchers for the first time, and promise to impact diverse fields beyond chromosome biology.